EP2857410A1 - Procédé de purification de 2´-fucosyllactose utilisant la chromatographie à lit mobile simulé - Google Patents

Procédé de purification de 2´-fucosyllactose utilisant la chromatographie à lit mobile simulé Download PDF

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Publication number
EP2857410A1
EP2857410A1 EP13187369.7A EP13187369A EP2857410A1 EP 2857410 A1 EP2857410 A1 EP 2857410A1 EP 13187369 A EP13187369 A EP 13187369A EP 2857410 A1 EP2857410 A1 EP 2857410A1
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Prior art keywords
fucosyllactose
solution
moving bed
process according
vol
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EP13187369.7A
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German (de)
English (en)
Inventor
Dr. Stefan Jennewein
Dr.Markus Helfrich
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Chr Hansen HMO GmbH
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Jennewein Biotechnologie GmbH
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Application filed by Jennewein Biotechnologie GmbH filed Critical Jennewein Biotechnologie GmbH
Priority to EP13187369.7A priority Critical patent/EP2857410A1/fr
Priority to DK14777665.2T priority patent/DK3063159T3/da
Priority to PL14777665T priority patent/PL3063159T3/pl
Priority to KR1020167008862A priority patent/KR102298281B1/ko
Priority to ES14777665T priority patent/ES2868249T3/es
Priority to PCT/EP2014/071145 priority patent/WO2015049331A1/fr
Priority to CN201811603666.0A priority patent/CN109705175B/zh
Priority to JP2016520025A priority patent/JP6666243B2/ja
Priority to US15/027,047 priority patent/US10435427B2/en
Priority to MX2016004288A priority patent/MX2016004288A/es
Priority to CN201480054230.4A priority patent/CN105814070A/zh
Priority to EP14777665.2A priority patent/EP3063159B1/fr
Priority to BR112016007342-8A priority patent/BR112016007342B1/pt
Priority to RU2016109548A priority patent/RU2685537C2/ru
Publication of EP2857410A1 publication Critical patent/EP2857410A1/fr
Priority to PH12016500594A priority patent/PH12016500594A1/en
Priority to MX2020004844A priority patent/MX2020004844A/es
Priority to AU2018267652A priority patent/AU2018267652B2/en
Priority to US16/440,100 priority patent/US11168105B2/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • C—CHEMISTRY; METALLURGY
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    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1814—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns recycling of the fraction to be distributed
    • B01D15/1821—Simulated moving beds
    • B01D15/185—Simulated moving beds characterized by the components to be separated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
    • B01D61/422—Electrodialysis
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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    • A23L33/16—Inorganic salts, minerals or trace elements
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    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/18—Iodine; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/16—Diafiltration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present application discloses a process for the purification of 2'-fucosyllactose produced by fermentation, enzymatic synthesis or chemical synthesis or combinations thereof.
  • the process uses simulated moving bed (SMB) chromatography which allows the continuous purification of large quantities of 2'-fucosyllactose at high purity.
  • SMB simulated moving bed
  • the presented process allows the provision of 2'-fucosyllactose free of noxious chemicals like e.g. trace amounts of heavy metals or organic solvents.
  • 2'-fucosyllactose may be obtained in solid form by spray drying. The provided 2'-fucosyllactose is very well-suited for use in food applications.
  • Human milk represents a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements.
  • the by far most predominant fraction is represented by carbohydrates, which can be further divided into lactose and more complex oligosaccharides. Whereas lactose is used as an energy source, the complex oligosaccharides are not metabolized by the infant.
  • the fraction of complex oligosaccharides accounts for up to 1/10 of the total carbohydrate fraction and consists of probably more than 150 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and thus cannot be found in large quantities in the milk of other mammals, like for example domesticated dairy animals.
  • gel-filtration chromatography For the purification of complex oligosaccharides, such as certain human milk oligosaccharides, gel-filtration chromatography is the method of choice until now.
  • the disadvantage of gel-filtration chromatography is that it cannot be efficiently scaled up and it is unsuitable for continuous operation.
  • gel-filtration chromatography is not economical and renders it impossible to provide certain human milk oligosaccharides - like 2'-fucosyllactose - in reasonably amounts and quality to use them in human food.
  • SMB chromatography has its roots in the petrochemical and mineral industries.
  • Today, SMB chromatography is used by the pharmaceutical industry for the separation of enantiomers out of racemic mixtures.
  • SMB chromatography has already been used for the separation of the monosaccharide fructose from fructose-glucose solutions and for the separation of the disaccharide sucrose from sugar beet or sugar cane syrups on large-scale.
  • SMB chromatography has not been used for the purification of a trisaccharide - like 2'-fucosyllactose - from fermentation, biocatalysis or chemical synthesis reactions yet.
  • Simulated moving bed (SMB) chromatography was developed as a continuous separation process analogous to continuous chemical separation processes such as rectification.
  • rectification a countercurrent is established between the liquid and the gaseous phase, which allows then the continuous application of feed and withdrawal of product(s).
  • counter-current chromatographic operations in theory should achieve separations superior than conventional cross-current operations.
  • chromatographic counter-current operations would require the mobile and stationary phases to move in opposite directions.
  • SMB chromatography was developed as a practical solution to the difficulties related to the concept of moving solid chromatography material in a continuous chromatographic separation process.
  • the classical SMB concept involves four different zones with four external applied streams: a feed stream containing the components to be separated, a desorbent or mobile phase stream, an extract and a raffinate stream (with the raffinate stream representing the less retained component(s).
  • These liquid streams divide the SMB system into four different zones (each zone or section can comprises one or more columns) with the following tasks: zone I is required for the regeneration of the solid phase, the purpose of zone II is the desorption of the less strongly desorbed material, the task of zone III is the adsorption of the strongly adsorbed material and finally the task of zone IV is the adsorption of the less adsorptive material.
  • zone I is required for the regeneration of the solid phase
  • zone II is the desorption of the less strongly desorbed material
  • the task of zone III is the adsorption of the strongly adsorbed material
  • the task of zone IV is the adsorption of the less adsorptive material.
  • zone I and IV serve for regeneration of the solid phase (regeneration zones) whereas zones II and III can be regarded as the actual separation zones of the system (separation zones).
  • the system contains (for the close loop operation) a recycling pump for the mobile phase (desorbent), passing the mobile phase through the fixed zones in one direction. Counter-current flow is then achieved by the periodical shifting and continuous supply or withdrawal of feed, desorbent, and products sequentially from one column to the next in the system.
  • open loop 3 zones systems can be used as well.
  • the 3 zones open loop systems are economic in case fresh solvent is rather inexpensive e.g. in case water or water/ethanol is used as mobile phase.
  • the regeneration of the liquid phase is no more needed, thus making zone IV obsolete.
  • the present invention provides a process for purification of 2'-fucosyllactose in a continuous manner from a crude solution containing 2'-fucosyllactose and contaminants, wherein the purity of 2'-fucosyllactose in the solution is ⁇ 80 %.
  • the process is characterized in that the crude solution is applied to at least one purification step using simulating moving bed chromatography. In this way, a purified solution containing 2'-fucosyllactose at a purity of ⁇ 80 % is provided.
  • 2'-fucosyllactose may be provided at a high purity, without heavy metal contaminants and in a continuous fashion.
  • large amounts of high quality 2'-fucosyllactose may be provided in a very convenient and economical way.
  • the inventive process also turned out to be highly stable even without a step of regeneration of the column material (e.g. cationic column material) used in the simulating moving bed chromatography step. In fact, the whole process can be stably and continuously operated for several months.
  • the purity of 2'-fucosyl lactose in the crude solution its ⁇ 70 %, ⁇ 60 %, ⁇ 50 %, ⁇ 40 %, ⁇ 30 %, ⁇ 20 %, ⁇ 10 % or ⁇ 5 % and/or the purified solution contains 2'-fucosyl lactose at a purity of ⁇ 80 %, preferably of ⁇ 90 %.
  • the at least one simulating moving bed chromatography step may have
  • At least one of the columns contains 0.1 to 5000 kg of cationic exchange resin, preferably 0.2 to 500 kg of cationic exchange resin, more preferably 0.5 to 50 kg of cationic exchange resin, most preferably 1.0 to 20 kg of cationic exchange resin.
  • upscaling of the amount of cationic exchange material, the flow rate in the different zones, the feed rate, the eluent flow rate and/or the switching time is possible.
  • the upscaling may be by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or all possible scaling factors in between said values.
  • a strong cationic exchange resin may be used as stationary phase.
  • the cation exchange resin is a sulfonic acid resin, more preferably a Purolite ® PCR833H (Purolite, Ratingen, Germany), Lewatit MDS 2368 and/or Lewatit MDS 1368 resin. If a cationic ion exchange resin is employed in the columns, it may be regenerated with sulphuric acid. Sulphuric acid can be employed in the eluent, preferably at concentration of 10 mM sulphuric acid or less.
  • the inventive is characterized in that the purified solution is applied to at least one further purification step using simulating moving bed chromatography, wherein a purified solution containing 2'-fucosyllactose at a purity of ⁇ 90 %, preferably ⁇ 92%; more preferably ⁇ 93% is provided.
  • the further simulating moving bed chromatography may have
  • At least one of the columns contains 0.1 to 5000 kg of cationic exchange resin, preferably 0.2 to 500 kg of cationic exchange resin, more preferably 0.5 to 50 kg of cationic exchange resin, most preferably 1.0 to 20 kg of cationic exchange resin.
  • upscaling of the amount of cationic exchange material, the flow rate in the different zones, the feed rate, the eluent flow rate and/or the switching time is possible.
  • the upscaling may be by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or all possible scaling factors in between said values.
  • the pH of the purified solution may be adjusted to pH 7, preferably by adding a base, more preferably by adding NaOH (e.g. 0.2 M NaOH).
  • the crude solution containing 2'-fucosyllactose and contaminants may be selected from the group consisting of microbial fermentation extract, biocatalysis reaction solution, chemical synthesis solution and combinations thereof. Fermentation as a source of 2'-fucosyllactose has the advantage that it is more cost-effective than chemical synthesis or biocatalysis i.e. enzymatic synthesis.
  • the solution containing 2'-fucosyl lactose is
  • the solution containing 2'-fucosyl-lactose is electrodialysed to a conductivity of ⁇ 15 mS/cm, preferably ⁇ 10 mS/cm, more preferably ⁇ 5 mS/cm and/or employing a membrane with a size exclusion limit of ⁇ 20 ⁇ .
  • the crude solution is electrodialysed before applying the solution to at least one purification step using simulating moving bed chromatography
  • major contaminants depend on the origin of the 2'-fucosyllactose fractions (i.e. chemical synthesis, biocatalysis or fermentation).
  • Typical contaminants are monosaccharides (e.g. glucose, galactose, fucose, etc.), disaccharides (e.g. lactose) and side products (e.g. lactulose).
  • the crude solution usually comprises the employed carbon source (e.g. glycerol, sucrose and/or glucose) as well as byproducts of the employed microbes (e.g. higher molecular mass oligosaccharides) as contaminants.
  • Said contaminants can efficiently be removed by a purification step using simulated moving bed (SMB) chromatography.
  • SMB simulated moving bed
  • the solution containing 2'-fucosyllactose may be concentrated, in particular
  • the purified solution is sterile filtered and/or subjected to endotoxin removal, preferably by filtration of the purified solution through a 3 kDa filter.
  • the purified solution may be spray-dried, particularly spray-dried at a 2'-fucosyllactose concentration of 20-60 (w/v), preferably 30-50 (w/v), more preferably 35-45 (w/v), an inlet temperature of 110-150 °C, preferably 120-140 °C, more preferably 125-135 °C and/or an exhaust temperature of 60-80 °C, preferably 65-70 °C.
  • the invention thus provides 2'-fucosyllactose which is producible with the process according to claim 11, wherein the 2'-fucosyllactose is preferably spray-dried.
  • the purified 2'-fucosyllactose has the advantage of being highly pure and being free of heavy metal contaminants, and/or organic solvents.
  • the 2'-fucosyllactose according to the invention can have
  • the 2'-fucosyllactose may be used in medicine, preferably in prophylaxis or therapy of gastrointestinal disorders. It can also be used in nutrition, preferably medicinal nutrition or dairy nutrition (e.g. cereal products).
  • 2'-fucosyllactose according to the invention as additive in food, preferably as additive in human food, more preferably as additive in human baby food.
  • Figure 1 schematically illustrates one purification step using simulating moving bed chromatography.
  • the simulating moving bed chromatography may have e.g. 12 columns in a serial arrangement 1, wherein the arrangement is divided in four different zones I, II, III and IV.
  • the crude solution containing 2'-fucosyllactose and contaminants is applied between zone II and III to the feed entry 2.
  • Extract is removed from exit 4 between zone I and zone II whereas raffinate is removed at exit 3 between zone III and zone IV.
  • Raffinate at exit 3 contains purified 2'-fucosyllactose
  • extract at exit 4 contains low molecular weight contaminants (e.g. monosaccharides and disaccharides).
  • FIG. 2 schematically illustrates two subsequent purification steps using simulating moving bed chromatography.
  • Each simulating moving bed chromatography may have e.g. 12 columns in a serial arrangement 1, 1', wherein each arrangement is divided in four different zones la, IIa, IIIa and IVa or zones Ib, IIb, IIIb and IVb, respectively.
  • the crude solution containing 2'-fucosyllactose and contaminants is applied between zone IIa and IIIa of the first arrangement 1 to the feed entry 2. Extract is removed at exit 4 between zone la and zone IIa whereas raffinate leaves the first serial arrangement 1 at exit 3 between zone IIIa and zone IVa and is applied to the second serial arrangement 1' between zone IIb and IIIb.
  • extract is removed at exit 6 whereas raffinate is removed at exit 5 between zone IIIb and zone IVb.
  • Raffinate at exit 5 contains highly purified 2'-fucosyllactose whereas extract as exit 6 contains high molecular weight contaminants (e.g. higher oligosaccharides).
  • FIG. 3 schematically illustrates a preferred purification scheme according to the present invention.
  • a solution 7 containing 2'-fucosyllactose and contaminants is applied to an electrodialysis step 8 until a conductivity of ⁇ 0.5 mS/cm is obtained.
  • Said solution is concentrated until the solution has reached a concentration of 2'-fucosyllactose of approx. 40% (w/v).
  • said solution is applied to at least one purification step using simulated moving bed chromatography 9.
  • a purified solution containing 2'-fucosyllactose with high purity is obtained.
  • Said pure solution is subjected to sterile filtration 11 (preferably also endotoxin removal).
  • an additional step of electrodialysis 10 with subsequent concentration may optionally be performed.
  • the purified solution containing 2'-fucosyllactose is subjected to spray drying 12 and pure, spray dried 2'-fucosyllactose 13 is obtained in solid granule form.
  • Figure 4 shows the result of an X-ray powder diffraction analysis of two samples of spray-dried 2'-fucosyllactose according to the present invention (sample #1 and sample #2).
  • the two obtained diffractograms reveal that both sample #1 and sample #2 are in the fully amorphous state (no characteristic peaks of crystalline matter).
  • Figure 5 shows the particle size distribution of spray-dried 2'-fucosyllactose according to the present invention (sample #1 and sample #2) determined by laser diffraction. A mean particle size of approx. 68 ⁇ m was determined for sample #1. Sample #2 had a mean particle size of approx. 44 ⁇ m. Both values are considered to be high for a spray-dried product.
  • Example 1 Purification of 2'-fucosyllactose using simulated moving bed chromatography (SMB chromatography).
  • a clear, particle-free solution containing 2'-fucosyllactose at a concentration of 250 g/L was electrodialysed to 0.5 mS/cm using a PC-Cell BED 1-3 electrodialysis apparatus (PC-Cell, Heusweiler, Germany) equipped with PC-Cell E200 membrane stack.
  • Said stack contained the following membranes: cation exchange membrane CEM: PC SK and the anion exchange membrane AEM:PcAcid60 having a size exclusion limit of 60 Da.
  • the 2'-fucosyllactose solution was concentrated to 300 g/L employing a vacuum concentrator at 40 °C.
  • a close loop SMB system equipped with 12 columns (Prosep ® columns with the dimensions: 40 mm x 740 mm (Latek, Eppelheim, Germany)) arranged in 4 zones was employed. Each column contained 760 g of Purolite ® PCR833H+(Purolite, Ratingen, Germany) strong cationic ion exchanger resin.
  • the system was operated at a temperature of 25°C with the following flow set parameters: flow rate zone I was 30.00 ml/min, flow rate zone II was set to 21.00 ml/min, flow rate zone III was 21,48 ml/min, flow rate of zone IV was set to 18.44 ml/min, feed was set to 3.00 ml/min, eluent flow was set to 11.56 ml/min and switching time was set to 17.92 min. As eluent, water with 10 % (v/v) food grade ethanol was used.
  • the 2'-fucosyllactose was marginally diluted through the SMB purification step - the concentration of 2'-fucosyllactose in the raffinate was determined with 200g/l.
  • the pH of the raffinate was adjusted to pH 7 by using 0.2 N NaOH. Under the described setting the SMB systems could be continuously operated for at least 3 months.
  • the obtained solution was again subjected to electrodialysis until a conductivity of less than 0.5 mS/cm was obtained and concentrated to obtain a 40% (w/v) 2'-fucosyllactose solution.
  • the solution was then subjected to sterile filtration and endotoxin removal by passing the solution through a 3 kDa filter (Pall Microza ultrafiltration hollow fiber module SEP-2013, Pall Corporation, Dreieich).
  • Example 2 Purification of 2'-fucosyllactose using multicomponent SMB chromatographic separation.
  • a clear, particle-free solution containing 2'-fucosyllactose at a concentration of 250g/L was electrodialysed to 0.5 mS/cm using a PC-Cell BED 1-3 electrodialysis apparatus (PC-Cell, Heusweiler, Germany) equipped with PC-Cell E200 membrane stack.
  • Said stack contained the following membranes: cation exchange membrane CEM:Pc SK and anion exchange membrane AEM:Pc Acid 60 which possess a size exclusion limit of 60 Da.
  • the 2'-fucosyllactose solution was concentrated to 300 g/L employing a vacuum concentrator at 40 °C.
  • a close loop multicomponent SMB System equipped with 24 columns (Prosep ® columns with the dimensions: 40 mm x 740 mm (Latek, Eppelheim, Germany)) arranged in 2 x 4 zones was employed. Each column contained 760 g of Purolite ® PCR833H+ (Purolite, Ratingen, Germany) strong cationic ion exchanger resin.
  • flow rate zone la was 30.00 ml/min
  • flow rate zone IIa was set to 21.00 ml/min
  • flow rate of zone IIIa was set to 21.48 ml/min
  • flow rate of zone IVa was set to 18.44 ml/min
  • feed was set to 3.00 ml/min
  • eluent flow was set to 11.56 ml/min
  • switching time was set to 17.92 min.
  • the raffinate of the first separation was passed on with a flow rate of 3.04 ml/min to a second separation step.
  • the flow rate of zone Ib was kept at 30 ml/min
  • flow rate of zone IIb was set to 19.61 ml/min
  • flow rate of zone IIIb was set to 21.63 ml/min
  • flow rate of zone IVb was set to 18.46 ml/min
  • eluent flow was set similarly to 11.56 ml/min and switching time of zones Ib to IVb was 10.46 min.
  • contaminants such as lactose, monosaccharides such as fucose, glucose, galactose and glycerol were found in the extract of the first separation step and larger oligosaccharide contaminants (e.g. difucosyllactose) were fractioned into the raffinate of the second separation step.
  • oligosaccharide contaminants e.g. difucosyllactose
  • 2'-fucosyllactose was fractionated into the raffinate of the first separation step and the extract of the second separation step and was thus free of low and high molecular weight contaminants. 2'-fucosyllactose was only marginally diluted through the SMB purification step - the concentration of 2'-fucosyllactose in the extract of the second purification step was determined with 200g/L.
  • the pH of the raffinate after the first separation step was adjusted to pH 7 by using 0.2 N NaOH.
  • Example 3 Obtaining 2'-fucosyllactose in solid form by spray drying.
  • the 2'-fucosyllactose fractions obtained by SMB chromatography were again subject to electrodialysis treatment until a conductivity of less than 0.5 mS/cm were obtained.
  • the fractions were then concentrated under vacuum to obtain 2'-fucosyllactose fractions containing 40% (w/v) 2'-fucosyllactose.
  • the solutions were subsequently subjected to sterile filtration and endotoxin removal by passing the solution through a 3 kDa filter (Pall Microza ultrafiltration hollow fiber module SEP-2013, Pall Corporation, Dreieich, Germany).
  • the so obtained sterile 2'-fucosyllactose solutions were then spray dried using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany).
  • a NUBILOSA LTC-GMP spray dryer NUBILOSA, Konstanz, Germany.
  • the 40% (w/v) solution was passed under pressure through the spray dryer nozzles with an inlet temperature set to 130°C. The flow was adjusted to maintaining an exhaust temperature between 66°C to 67°C.
  • DSC Differential scanning calorimetry
  • a Mettler Toledo 821e (Mettler Toledo, Giessen, Germany) was used to determine thermal events of two samples (sample #1 and sample #2) of spray-dried 2'-fucosyllactose.
  • Table 1 sample 1 st heating scan 2 nd heating scan Tg endotherm exotherm Tg1 Tg2 exotherm °C °C J/g °C J/g °C °C J/g #1 63.9 87.9 -0.5 82.8 0.7 67.4 122.6 n.d. n.d. #2 87.1 n.d. n.d. n.d. n.d. 84.6 n.d. 125.9 1.1 n.d.: not detected
  • DSC analysis of the sample #1 revealed a main glass transition (Tg) at 67.4 °C in the 2 nd heating scan.
  • Tg main glass transition
  • a small second Tg was also detected at 122.6 °C in the 2 nd heating scan.
  • the main glass transition was detected in the first 1 st heating scan followed by an exo- and endothermic event at temperatures above the Tg. These events are attributed to relaxation effects in the sample.
  • XRD Wide angle X-ray powder diffraction
  • Laser-diffraction measurements were performed using a Partica LA-950 Laser Diffraction Particle Size Distribution Analyzer (Horiba, Kyoto, Japan) equipped with a 605 nm laser diode for detecting particles >500 nm and 405 nm blue light emitting diode (LED) for detecting particles ⁇ 500 nm.
  • Isooctane was used as dispersion medium (refractive index of 1.391). Since the refractive index of the samples was unknown, the refractive index of sugar (disaccharide) particles was used (1.530).
  • the samples were dispersed in isooctane by ultrasonication for up to 5 minutes. Prior to measurement, the system was blanked with isooctane. The dispersion of each sample was measured 3 times and the mean values and the standard deviation are reported.
  • the mean particle size (weighted average of particle sizes by volume) and the mode particle size (peak of the distribution) are reported. In addition to the particle distribution by volume (q%), the result are reported as:
  • sample #1 and #2 The particle size distribution for sample #1 and #2 is shown in Figure 5 .
  • the mode size which represents the particle size of the highest intensity, is comparable for both samples.
  • the mean particle size of 67.85 ⁇ m (sample #1) and 43.65 ⁇ m (sample #2), respectively, is regarded as unusually high for spray-dried particles.
  • the fraction detected at higher particle diameters is probably caused by agglomerated powder particles.

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CN201480054230.4A CN105814070A (zh) 2013-10-04 2014-10-02 使用模拟移动床色谱纯化中性人乳寡糖的方法
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EP3063159A1 (fr) 2016-09-07
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WO2015049331A1 (fr) 2015-04-09
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